The central hypothesis of this Project 1 is that CCR5-deficient, C04-)- T-cells played a major role in contributing to the successful outcome of the Berlin patient. Our approach is tiased on our expertise with cell'based therapies for HIV infection as well as the first in-human data from Infusions of autologous CD4 cells rendered CCRS-deficient by zinc finger nucleases (ZFNs), whk:h bind to, cleave and Inactivate the ccrS gene. Phase-I data from this trial demonstrated an excellent safety profile as well as efficient, long-temn engraftment and expansion of the CCRS-modified cells. Furthermore, while not a direct goal of this trial, intriguing antiviral effects have been observed in 6 subjects at Penn who completed a 12 week treatment interruption, in this project we will detennine if patient conditioning with a single dose of cyclophosphamide that is used routinely in autoimmune disorders and to promote immunotherapy can enhance engraftment of autologous T-cells treated with RS-spedfic ZFNs, and as a result, control HIV In the absence of H/\/^T. Three specific aims are proposed: (1) Complete the pre-clinical testing necessary to support the manufacturing of mRNA ZFN modified CD4 T cells; (2) Conduct a proof of concept clinical trial to detennine the safety of R5 deficient CD4 cells infused in the setting of transient lymphopenia, induced with a single dose of cyclophosphamide and (3) Evaluate the host and virotogical response to R5-modified T ceil infusions. We hypothesize that the level of engraftment will increase in the cyclophosphamide-treated patients, and related to this, ttiat antiviral effects will be uncovered during a structured treatment interruption. Curing or controlling HIV in the absence of HAART has been an elusive goal, but one that may finally be realized. The results of our project, conducted with an experienced team in place at Sangamo and Penn will provide critical information for future protocols to further dtesect the factors that led to cure of the Beriin patient and provide basic insights into underiying mechanisms.